Joan M. Robbins

Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).

Phase 1 trial of vocimagene amiretrorepvec and 5-fluorocytosine for recurrent high-grade glioma

Toca 511 and Toca FC show promising results in treating recurrent high-grade glioma, and a specific molecular signature correlates with treatment-related survival. Tag-team attack on glioma Toca FC (extended-release 5-fluorocytosine) and Toca 511 (vocimagene amiretrorepvec) are an investigational therapeutic combination for glioma, consisting of two parts: a prodrug that is inactive on its own and a modified virus that infects the tumor and delivers an enzyme, which then activates the drug and allows it to kill the glioma cells. Cloughesy et al. tested this therapy in 45 human patients with recurrent or progressive high-grade glioma and discovered that the treatment was well tolerated and improved survival compared to an external control group. In addition, the authors identified a gene signature that correlated with response to the treatment, which may help identify the patients most likely to benefit from this approach. Toca 511 (vocimagene amiretrorepvec) is an investigational nonlytic, retroviral replicating vector (RRV) that delivers a yeast cytosine deaminase, which converts subsequently administered courses of the investigational prodrug Toca FC (extended-release 5-fluorocytosine) into the antimetabolite 5-fluorouracil. Forty-five subjects with recurrent or progressive high-grade glioma were treated. The end points of this phase 1, open-label, ascending dose, multicenter trial included safety, efficacy, and molecular profiling; survival was compared to a matching subgroup from an external control. Overall survival for recurrent high-grade glioma was 13.6 months (95% confidence interval, 10.8 to 20.0) and was statistically improved relative to an external control (hazard ratio, 0.45; P = 0.003). Tumor samples from subjects surviving more than 52 weeks after Toca 511 delivery disproportionately displayed a survival-related mRNA expression signature, identifying a potential molecular signature that may correlate with treatment-related survival rather than being prognostic. Toca 511 and Toca FC show excellent tolerability, with RRV persisting in the tumor and RRV control systemically. The favorable assessment of Toca 511 and Toca FC supports confirmation in a randomized phase 2/3 trial (NCT02414165).

Phase I trial of interferon-γ (IFN-γ) retroviral vector administered intratumorally to patients with metastatic melanoma

Background: Interferon-γ (IFN-γ) gene/retroviral vector cell vaccinations have generated protective responses from unmodified tumor cell challenges as well as a regression of established tumors in animal models. The purpose of this trial was to determine the feasibility and safety of a direct intratumoral injection of IFN-γ retroviral vector in advanced melanoma patients.Methods: This was a phase I study, in which 13 patients received a single daily injection of a retroviral vector with the IFN-γ gene for 5 consecutive days (1.5 × 108 colony-forming units total dose); patients subsequently underwent resection of the injected lesion to confirm DNA transduction in situ.Results: No toxicity related to the injected vector was observed. Replication competent retrovirus was not observed in any prepared samples (n = 65). IFN-γ expression was confirmed in 3 of 10 harvested tumor samples; one was equivocal, and DNA transduction was unable to be confirmed by enzyme-linked immunospot assay in six samples.Conclusions: An injection of IFN-γ gene/retroviral vector is well tolerated. DNA transduction was demonstrated in human subjects, confirming the feasibility of the direct injection approach for the gene therapy of solid tumors. Further trials to determine optimal schedule and potential efficacy are indicated.

Toca 511 gene transfer and 5-fluorocytosine in combination with temozolomide demonstrates synergistic therapeutic efficacy in a temozolomide-sensitive glioblastoma model

Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized cytosine deaminase (CD) gene to tumors in orthotopic glioma models. This agent, in conjunction with subsequent oral extended-release 5-fluorocytosine (5-FC) (Toca FC), is currently under investigation in patients with recurrent high-grade glioma . Temozolomide (TMZ) with radiation is the most frequently used first-line treatment for patients with glioblastoma, the most common and aggressive form of primary brain cancer in adults. However, subsets of patients with certain genetic alterations do not respond well to TMZ treatment and the overall median survival for patients who respond remains modest, suggesting that combinatorial approaches may be necessary to significantly improve outcomes. We show that in vitro TMZ delays but does not prevent RRV spread, nor interfere with Toca 511+5-FC-mediated cell killing in glioma tumor cells, and in vivo there is no significant hematologic effect from the combination of 5-FC and the clinically relevant dose of TMZ. A synergistic long-term survival advantage is observed in mice bearing an orthotopic TMZ-sensitive glioma after Toca 511 administration followed by coadministration of TMZ and 5-FC. These results provide support for the investigation of this novel combination treatment strategy in patients with newly diagnosed malignant glioma.

Intravenous Administration of Retroviral Replicating Vector, Toca 511, Demonstrates Therapeutic Efficacy in Orthotopic Immune-Competent Mouse Glioma Model

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.

Convection-enhanced delivery improves distribution and efficacy of tumor-selective retroviral replicating vectors in a rodent brain tumor model

In the present study, we compared the therapeutic effect of tumor-selective retroviral replicating vectors (RRV) expressing the yeast cytosine deaminase (CD) delivered by convection-enhanced delivery (CED) or simple injection, followed by systemic administration of the pro-drug, 5-fluorocytosine (5-FC). Treatment with RRV-CD and systemic 5-FC significantly increased survival in rodent U87MG glioma model in comparison with controls (P<0.01). Interestingly, CED of RRV-CD followed by 5-FC further enhanced survival in this animal model in comparison with intra-tumoral injection of RRV-CD, followed by systemic 5-FC (P<0.05). High expression levels of Ki-67 were found in untreated tumors compared with treated. Untreated tumors were also much larger than treated. CED resulted in excellent distribution of RRV while only partial distribution of RRV was obtained after injection. Furthermore, RRV-CD and CD were also found in tumors from treated rats at study end points. These results demonstrated that RRV vectors may efficiently transduce and stably propagate in malignant human glioma, thereby achieving a significant in situ amplification effect after initial administration. We conclude that delivery of RRV into the glioma by CED provides much wider vector distribution than simple injection, and this correlated with better therapeutic outcomes.

Retroviral replicating vectors in cancer.

The use of replication-competent viruses for the treatment of cancer is an emerging technology that shows significant promise. Among the various different types of viruses currently being developed as oncolytic agents, retroviral replicating vectors (RRVs) possess unique characteristics that allow highly efficient, non-lytic, and tumor-selective gene transfer. By retaining all of the elements necessary for viral replication, RRVs are capable of transmitting genes via exponential in situ amplification. Their replication-competence also provides a powerful means whereby novel and useful RRV variants can be generated using natural selection. Their stringent requirement for cell division in order to achieve productive infection, and their preferential replication in cells with defective innate immunity, confer a considerable degree of natural specificity for tumors. Furthermore, their ability to integrate stably into the genome of cancer cells, without immediate cytolysis, contributes to long-lasting therapeutic efficacy. Thus, RRVs show much promise as therapeutic agents for cancer and are currently being tested in the clinic. Here we describe experimental methods for their production and quantitation, for adaptive evolution and natural selection to develop novel or improved RRV, and for in vitro and in vivo assessment of the therapeutic efficacy of RRVs carrying prodrug activator genes for treatment of cancer.

Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.

Maintaining therapeutic activity in the operating room: compatibility of a gamma-retroviral replicating vector with clinical materials and biofluids

Toca 511 is a novel retroviral replicating vector, encoding a modified yeast cytosine deaminase, administered to recurrent high grade glioma patients in Phase 1 trials by stereotactic, transcranial injection into the tumor or into the walls of the resection cavity. A key issue, with little published data, is vector biocompatibility with agents likely to be encountered in a neurosurgical setting. We tested biocompatibility of Toca 511 with: delivery devices; MRI contrast agents, including ProHance supporting coinjection for real time MRI-guided intratumoral delivery; hemostatic agents; biofluids (blood and cerebrospinal fluid); potential adjuvants; and a needleless vial adapter that reduces risk of accidental needle sticks. Toca 511 is stable upon thawing at ambient temperature for at least 6 hours, allowing sufficient time for administration, and its viability is not reduced in the presence of: stainless steel and silica-based delivery devices; the potential MRI contrast agent, Feraheme; ProHance at several concentrations; the hemostatic agent SURGIFOAM; blood; cerebrospinal fluid; and the needleless vial adapter. Toca 511 is not compatible with the hemostatic agent SURGICEL or with extended exposures to titanium-based biopsy needles.

Abstract B33: Intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a novel prodrug activator gene transfer technology in an immune-competent model of glioblastoma

Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have poor prognosis and current treatments remain only palliative. Previous gene therapy approaches using replication-deficient viral vectors have demonstrated limited efficacy likely due to poor gene delivery to the tumor. Methods: We used a novel replication-competent retroviral vector (Toca 511) designed and engineered to efficiently deliver a modified cytosine deaminase (CD) prodrug activating gene to glioma cells. Previous studies showed that such a replicating vector can preferentially infect intracranial human xenografts in nude mice (Tai et al. Mol Ther. 2005;12:842) and lead to increased survival in treatment groups compared to controls. In preparation for clinical trials we have improved the CD gene activity and further stabilized the vector structure to create Toca 511. We investigated the functionality of Toca 511 using stereotactic, intratumoral delivery in a syngeneic, orthotopic glioma model in immune-competent B6C3F1 mice. Freshly excised tumors were analyzed by quantitative PCR, Western, and HPLC. Results: Brain tumors isolated from mice treated with Toca 511 and fluorocytosine (5FC) had observable expression of CD protein and vector copy numbers per genome that demonstrate efficient tumor infection. Extensive tumor specific viral spread with almost no spread of vector from the site of injection was observed in the first 24 days. Highly efficient intratumoral conversion of the 5FC prodrug into the anticancer drug 5-fluorouracil (5FU) was demonstrated after intraperitoneal or oral dosing with 5FC. Studies with Toca 511 at high and mid dose levels in combination with 5FC resulted in prolonged survival compared to controls (> 5 months, p Conclusions: Toca 511 efficiently delivers a functional CD gene for expression in glioma cells in a syngeneic, orthotopic model in immune-competent mice. Toca 511 increases survival through the delivery of CD gene and subsequent efficient conversion of 5FC into 5FU within the brain tumor with no safety related findings. A phase 1 ascending dose trial investigating the safety and tolerability of Toca 511 in combination with 5FC in patients with recurrent GBM is about to initiate. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B33.